The production of genetically engineered animals offers the potential for tremendous advances in the production of valuable animals having defined genetic characteristics and for the production of valuable pharmaceutical products from the cells of such animals. However, the production of genetically modified animals involves significant technical hurdles that have only been overcome for a few species. The ability to incorporate genetic modifications into the permanent DNA of a species requires several distinct technologies that must be developed for each genetically engineered species. One approach to alter the genetic and physical characteristics of an animal using embryonic stem cells that can contribute to the phenotype of an animal when injected into an embryonic form of the animal. Embryonic stem cells have the ability to contribute to the tissue of a chimeric animal born from the recipient embryo and to contribute to the genome of a transgenic organism created by breeding chimeras.
Significant expenditure of time and resources has been committed to the study and development of embryonic stem cell lines and culture techniques that permit such cells to be maintained in culture, particularly where known genetic engineering techniques can be applied to modify the genome of the embryonic stem cells. Although significant expenditures have been made, the ability to sustain the pluripotency of embryonic stem cells in culture has been achieved for only a few species, notably mice. For other species, the promise of genetic engineering has been frustrated by the lack of sustainable long-term embryonic stem cell cultures.
If cultures of embryonic stem cells were readily available, a broad application of new technologies would be available. Because embryonic stem cells contribute to the permanent DNA of an animal, the physiological characteristics of the animal from which an embryonic stem cell was derived can be transferred to a recipient embryo by incorporating these cells into the recipient animal in an embryonic state. This offers two principal advantages: phenotype of animal from which embryonic stem cells are derived can be selectively transferred to a recipient embryo. Second, when the embryonic stem cell cultures are particularly stable in, the cells can be modified genetically to introduce genetic modifications into a recipient embryo in which the embryonic stem cells are introduced.
In certain cases, the embryonic stem cells can be engineered with a transgene that encodes an exogenous protein. Thus, one particularly attractive field of research and attractive area for commercial development is genetically engineered animals that express exogenous proteins in their tissues. The ability to produce exogenous proteins in cells of an animal is a particularly valuable capability when tissue specific expression is obtained.
Assuming that the embryonic stem cell culture is sufficiently stable to allow a transgene to become integrated into the genome of the embryonic stem cell, the transgene encoding the protein can be passed to a new chimeric or transgenic organism by several different techniques depending on the specific construct used as the transgene. Whole genomes can be transferred by cell hybridization, intact chromosomes by microcells, subchromosomal segments by chromosome mediated gene transfer, and DNA fragments in the kilobase range by DNA mediated gene transfer (Klobutcher, L. A. and F. H. Ruddle, Annu. Rev. Biochem., 50: 533–554, 1981). Intact chromosomes may be transferred to an embryonic stem cell by microcell-mediated chromosome transfer (MMCT) (Fournier, R. E. and F. H. Ruddle, Proc. Natl. Acad. Sci. U.S.A., 74: 319–323, 1977).
As noted above, the performance of genetic modifications in embryonic stem cells to produce transgenic animals has been demonstrated in only a very few species. For mice, the separate use of homologous recombination followed by chromosome transfer to embryonic stem (ES) cells for the production of chimeric and transgenic offspring is well known. Powerful techniques of site-specific homologous recombination or gene targeting have been developed (see Thomas, K. R. and M. R. Capecchi, Cell 51: 503–512, 1987; review by Waldman, A. S., Crit. Rev. Oncol. Hematol. 12: 49–64, 1992). Insertion of cloned DNA (Jakobovits, A., Curr. Biol. 4: 761–763, 1994), and manipulation and selection of chromosome fragments by the Cre-loxP system techniques (see Smith, A. J. et al., Nat. Genet. 9: 376–385, 1995; Ramirez-Solis, R. et al., Nature 378: 720–724, 1995; U.S. Pat. Nos. 4,959,317; 6,130,364; 6,091,001; 5,985,614) are available for the manipulation and transfer of genes into murine ES cells to produce stable genetic chimeras. Many such techniques that have proved useful in mammalian systems would be available to be applied to non-mammalian embryonic stem cells if the necessary cultures were available.
Embryonic stem cell lines suitable for use in transgenesis must be stable and maintain pluripotency when the ES cell is transfected with a genetic construct, when the genetic construct is expressed in the ES cell to allow selection of successfully transformed cells, and during the injection into recipient embryos and the formation of resulting chimeras. Moreover, the somatic tissue of the chimera must exhibit the genetic modifications derived from the embryonic stem cells, and the genetic modification must be identified in the tissue of the chimeric animal. Ideally, the embryonic stem cell could be modified to contain a transgene that would not only be incorporated into the somatic tissue of a chimeric animal, but could be effectively expressed in a wide variety of tissues in the animal, specifically individual tissue types in which the transgene is designed to be expressed. For example, transgenes encoding DNA derived from the lymphoid elements of the immune system might be targeted to be expressed in B lymphocytes of a chimeric or transgenic animal. In such circumstances, the embryonic stem cell culture must allow transformation of the genome of the embryonic stem cell with a transgene containing DNA encoding an exogenous protein, and the embryonic stem cell must contribute significantly to the genome of the resulting animal.
Avian biological systems offer many advantages including efficient farm cultivation, rapid growth, and economical production. Globally, chickens and turkeys are a major source of protein in the human diet. Further, the avian egg offers an ideal biological design, both for massive synthesis of a few proteins and ease of isolation of protein product. However, application of the full range of mammalian transgenic techniques to avian species has been unsuccessful. Most notably, the transmission to a mature, living animal of a genetic modification introduced into an avian embryonic stem cell has not been demonstrated.
To conduct the types of transgenesis in avians that has been conducted in mice requires the development of avian embryonic stem cells, which can exist in culture and maintain their pluripotential capability, i.e. express an embryonic stem cell phenotype, for an extended period of time. In many cases, the techniques necessary to introduce genetic modifications into embryonic stem cells, the screening of modified embryonic stem cells to select specific cell modifications in which the genetic constructs have been introduced, and the ability to manipulate the ES cells for injection into embryos to produce transgenic chickens, requires at least several weeks for all of the steps to be performed. In order for the embryonic stem cells to be useful in transgenesis, the pluripotential state must be maintained for the entire time period up until injection into an embryo and the ES cell must be incorporated into a recipient embryo to a substantial degree to be detected in a resulting chimeric animal.
Unless the ES cell culture conditions are ideal, embryonic stem cells begin to differentiate in a short period of time and lose the ability to contribute to the somatic tissue of a chimeric organism derived from an embryo in which the cells are injected. Thus, when differentiation occurs, cells in culture are no longer useful as pluripotential cells and also cannot be used for transgenesis. Using current avian ES cell culture techniques, the short time periods during which ES cells maintain pluripotency in culture limits their use in creating chimeras and prevents the ability to create desirable chimeric or transgenic avians, specifically those expressing exogenous proteins.